Survivin minigene DNA vaccination is effective against neuroblastoma

The inhibitor of apoptosis protein survivin is highly expressed in neuroblastoma (NB) and survivin‐specific T cells were identified in Stage 4 patients. Therefore, we generated a novel survivin minigene DNA vaccine (pUS‐high) encoding exclusively for survivin‐derived peptides with superior MHC class I (H2‐K^k) binding affinities and tested its efficacy to suppress tumor growth and metastases in a syngeneic NB mouse model. Vaccination was performed by oral gavage of attenuated Salmonella typhimurium SL7207 carrying pUS‐high. Mice receiving the pUS‐high in the prophylactic setting presented a 48–52% reduction in s.c. tumor volume, weight and liver metastasis level in contrast to empty vector controls. This response was as effective as a survivin full‐length vaccine and was associated with an increased target cell lysis, increased presence of CD8⁺ T‐cells at the primary tumor site and enhanced production of proinflammatory cytokines by systemic CD8⁺ T cells. Furthermore, depletion of CD8⁺ but not CD4⁺ T‐cells completely abrogated the pUS‐high mediated primary tumor growth suppression, demonstrating a CD8⁺ T‐cell mediated effect. Therapeutic vaccination with pUS‐high led to complete NB eradication in over 50% of immunized mice and surviving mice showed an over 80% reduction in primary tumor growth upon rechallenge in contrast to controls. In summary, survivin‐based DNA vaccination is effective against NB and the rational minigene design provides a promising approach to circumvent potentially hazardous effects of using full length antiapoptotic genes as DNA vaccines. © 2009 UICC     

Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion, and promotion of tumor cell resistance to apoptosis. In our previous studies using non-small cell lung cancer (NSCLC) cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations, we demonstrated that constitutive overexpression of COX-2 leads to stabilization of the inhibitor of apoptosis protein survivin resulting in the elevated apoptosis resistance of COX-2-overexpressing cells. Genetic or pharmacologic suppression of COX-2 activity increased proteasomal degradation of survivin and cellular response to apoptosis induction. Our data show that expression of survivin in non-small cell lung cancer cells can be significantly down-regulated by RNA interference. Whereas COX-2-overexpressing NSCLC cells have significantly higher apoptosis resistance than the parental cells, inhibition of survivin expression by small interfering RNA decreases apoptosis resistance to the level of the parental non-small cell lung cancer. We conclude that COX-2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.     

Therapeutic targeting of the survivin pathway in cancer: initiation of mitochondrial apoptosis and suppression of tumor-associated angiogenesis.

PURPOSE: Molecular antagonists of the inhibitor of apoptosis protein survivin have shown promise as novel anticancer strategies for triggering tumor cell apoptosis, dysregulating mitotic progression, and inhibiting tumor growth in preclinical models. However, how survivin couples to the cell death machinery has remained elusive, and the relevant cellular targets of survivin antagonists have not been completely elucidated. Experimental Design: Human umbilical vein and dermal microvascular endothelial cells were infected with replication-deficient adenoviruses encoding survivin (pAd-Survivin), green fluorescent protein (pAd-GFP), or a phosphorylation-defective survivin Thr(34)-->Ala (pAd-T34A) dominant negative mutant. The effect of wild-type or mutant survivin was investigated on capillary network stability, endothelial cell viability, and caspase activation in vitro and on kinetics of tumor growth and development of angiogenesis in a breast cancer xenograft model in vivo. The cell death pathway initiated by survivin targeting was mapped with respect to cytochrome c release, changes in mitochondrial transmembrane potential, and apoptosome requirements using mouse embryonic fibroblasts deficient in Apaf-1 or caspase-9. RESULTS: Adenoviral transduction of endothelial cells with pAd-Survivin inhibited growth factor deprivation- or ceramide-induced apoptosis, reduced caspase-3 and -7 generation, and stabilized three-dimensional capillary networks in vitro. Conversely, expression of pAd-T34A caused apoptosis in umbilical vein and dermal microvascular endothelial cells and resulted in caspase-3 activity. Cell death induced by survivin targeting exhibited the hallmarks of mitochondrial-dependent apoptosis with release of cytochrome c and loss of mitochondrial transmembrane potential and was suppressed in Apaf-1 or caspase-9 knockout mouse embryonic fibroblasts. When injected in human breast cancer xenografts, pAd-T34A inhibited growth of established tumors and triggered tumor cell apoptosis in vivo. This was associated with a approximately 60% reduction in tumor-derived blood vessels by quantitative morphometry of CD31-stained tumor areas, and appearance of endothelial cell apoptosis by internucleosomal DNA fragmentation in vivo. CONCLUSIONS: Survivin functions as a novel upstream regulator of mitochondrial-dependent apoptosis, and molecular targeting of this pathway results in anticancer activity via a dual mechanism of induction of tumor cell apoptosis and suppression of angiogenesis.     

Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer

Survivin plays an important role in cancer development. We aim to show here that suppression of survivin expression or function by antisense and dominant-negative (DN) mutant can inhibit gastric cancer carcinogenesis and angiogenesis in vivo. Plasmid constructs expressing survivin antisense and DN mutant replacing the cysteine residue at amino acid 84 with alanine (Cys84Ala) were prepared and introduced into BCG-823 and MKN-45 gastric cancer cells to establish stable transfectants. We showed that both antisense and DN mutant stable transfectants exhibited abnormal morphology, with decreased cell growth and increased rate of spontaneous apoptosis and mitotic catastrophe. Furthermore, in nude mice xenografts, these cells exhibited decreased de novo gastric tumor formation and reduced development of angiogenesis. Results from these studies strongly suggest that survivin is a promising target for gastric cancer treatment.     

YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts

Various accumulating evidence suggests that survivin, a member of the inhibitor of apoptosis (IAP) family, plays an important role in drug resistance and cancer cell survival in many types of cancer, including hormone-refractory prostate cancer (HRPC). Here, we characterized YM155, a novel small-molecule survivin suppressant, using a survivin gene promoter activity assay. YM155 suppressed expression of survivin and induced apoptosis in PC-3 and PPC-1 human HRPC cell lines at 10 nmol/L. In contrast, YM155 up to 100 nmol/L showed little effect on expression levels of other IAP- or Bcl-2-related proteins. In a s.c. xenografted PC-3 tumor model in mice, 3-day continuous infusions of YM155 at 3 to 10 mg/kg induced massive tumor regression accompanied by suppression of intratumoral survivin. YM155 also completely inhibited the growth of orthotopically xenografted PC-3 tumors. No significant decreases in body weight were observed in mice treated with YM155 during the experimental period. Pharmacokinetic analyses indicated that YM155 is highly distributed to tumors and at concentrations approximately 20-fold higher than those in plasma. Our findings represent the first attempt to show tumor regression and suppression of survivin in p53-deficient human HRPC cells by a single small molecular compound treatment. Further extensive investigation of YM155 in many types of cancer, including HRPC, seems to be worthwhile to develop this novel therapeutic approach.     

Survivin knockdown and concurrent 4-HPR treatment controlled human glioblastoma in vitro and in vivo

Survivin is highly expressed in most cancers, including glioblastoma, and it plays a significant role in inhibiting apoptosis and promoting tumor growth. Treatment of cancer cells with N-(4-hydroxyphenyl) retinamide (4-HPR) induces apoptosis through destabilization of mitochondrial membrane and activation of caspase-mediated apoptotic pathways. We studied the efficacy of a combination of survivin knockdown and 4-HPR treatment to induce apoptosis and inhibit invasion, angiogenesis, and growth of human glioblastomas in vitro and in vivo. Using a plasmid encoding survivin shRNA, we downregulated survivin in glioblastoma U251MG and U118MG cells and simultaneously treated with 1 µM 4-HPR for 48 hours. Cells following treatments were subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and invasion assays. In vivo angiogenesis and tumor regression studies were performed in nude mice. TUNEL assay demonstrated apoptosis in more than 80% of cells after survivin knockdown and 4-HPR treatment. Matrigel invasion assays demonstrated marked decreases in tumor cell invasion. In vivo angiogenesis studies depicted a remarkable inhibition of neovascularization due to the knockdown of survivin and 4-HPR treatment. Imaging of intracerebral tumorigenesis and longitudinal studies on subcutaneous solid tumor formation showed dramatic decreases in tumorigenesis and solid tumor progression, respectively, after treatment with the combination. Studies to elucidate the molecular mechanisms of the inhibition of angiogenesis and tumor regression demonstrated marked decreases in proliferating cell nuclear antigen, metalloproteinase-9, vascular endothelial growth factor, basic fibroblast growth factor, and CD31 in solid tumors. Our data demonstrated that survivin knockdown and concurrent 4-HPR treatment could be a novel therapeutic strategy for controlling growth of human glioblastomas.     

Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol

Survivin is a member of the inhibitor of apoptosis proteins that is expressed at high levels in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Naturally occurring dietary compounds such as resveratrol have gained considerable attention as cancer chemopreventive agents. Here, we discovered a novel function of the chemopreventive agent resveratrol: resveratrol is a potent sensitizer of tumor cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through p53-independent induction of p21 and p21-mediated cell cycle arrest associated with survivin depletion. Concomitant analysis of cell cycle, survivin expression, and apoptosis revealed that resveratrol-induced G(1) arrest was associated with down-regulation of survivin expression and sensitization for TRAIL-induced apoptosis. Accordingly, G(1) arrest using the cell cycle inhibitor mimosine or induced by p21 overexpression reduced survivin expression and sensitized cells for TRAIL treatment. Likewise, resveratrol-mediated cell cycle arrest followed by survivin depletion and sensitization for TRAIL was impaired in p21- deficient cells. Also, down-regulation of survivin using survivin antisense oligonucleotides sensitized cells for TRAIL-induced apoptosis. Importantly, resveratrol sensitized various tumor cell lines, but not normal human fibroblasts, for apoptosis induced by death receptor ligation or anticancer drugs. Thus, this combined sensitizer (resveratrol)/inducer (e.g., TRAIL) strategy may be a novel approach to enhance the efficacy of TRAIL-based therapies in a variety of human cancers.     

糖化重组生存素腺病毒疫苗抗肿瘤作用的实验研究

背景与目的：生存素在调控细胞凋亡和有丝分裂过程中起着重要作用,在正常分化的组织中不表达或表达极低,而在胚胎组织和大部分肿瘤组织中广泛表达,而这一特点为恶性肿瘤的基因治疗提供了很好的靶标.本研究制备糖化重组生存素腺病毒疫苗,观察其抗肿瘤作用并探讨其作用机制.方法：将重组生存素腺病毒和甘露聚糖在氧化条件下偶联,同时建立小鼠结肠癌和Lewis肺癌模型,以糖化重组生存素腺病毒疫苗治疗荷瘤小鼠,观察肿瘤体积和小鼠生存时间.对小鼠肿瘤组织作TUNEL染色观察肿瘤细胞凋亡.为了检测疫苗的保护作用,预先以糖化重组生存素腺病毒疫苗免疫接种小鼠,或合用抗CD4、CD8和NK单克隆抗体阻断相应的淋巴细胞亚群,再接种肿瘤,观察小鼠肿瘤生长情况.结果：糖化重组生存素腺病毒疫苗治疗组小鼠肿瘤生长受到明显抑制,无明显不良反应发生,小鼠的生存时间明显延长,肿瘤细胞凋亡明显增加.同时,糖化重组生存素腺病毒疫苗能诱导小鼠产生抗肿瘤免疫,这一免疫反应依赖于CD4和CD8淋巴细胞,也部分依赖于NK细胞.结论：糖化重组生存素腺病毒疫苗能诱导机体产生特异性的主动免疫反应,诱导肿瘤细胞凋亡,具有明确的抗肿瘤作用,值得进一步研究.     

Reduced mammary tumor progression in WAP-TAg/WAP-maspin bitransgenic mice

Maspin is a unique serpin involved in the suppression of tumor growth and metastasis. To investigate whether increased levels of maspin protect against tumor progression in vivo, we established a transgenic model in which maspin is targeted to mammary epithelial cells by the Whey Acidic Protein (WAP) promoter for overexpression. We crossed these WAP-maspin transgenic mice with the WAP-TAg mouse model of tumor progression. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. The number of pulmonary metastases was reduced in the presence of maspin overexpression. These data demonstrate that targeted overexpression of maspin can inhibit tumor progression in vivo, likely through a combination of increased apoptosis, decreased angiogenesis, and inhibition of tumor cell migration.     

p53 induction and apoptosis in response to radio- and chemotherapy in vivo is tumor-type-dependent

The p53 protein rapidly accumulates in cells in response to DNA damage, which can trigger apoptosis. This pathway is hypothesized to be important for tumor suppression by p53, as well as for the response of tumors to chemo- or radiotherapy. Implicit in these ideas is that the p53 induction-apoptosis pathway is active in tumor cells in vivo. Because tumor suppression by p53 in mice is markedly tissue-type-dependent, we tested the activity of the pathway in tumors in vivo by inducing tumors in six different tissues and treating tumor-bearing mice with DNA damaging cancer therapeutic agents. In response to treatment, cells from T-cell lymphomas, intestinal adenomas, and mammary tumors rapidly induced p53 and underwent apoptosis. In squamous cell papillomas, p53 was constitutively expressed and was further induced by the treatments, but apoptotic cells were only rarely observed. In treated mice bearing lung or liver adenomas, minimal or no p53 accumulation or apoptosis was observed in the tumor cells. Thus, there is marked variation in the intrinsic ability of autochthonous tumor cells to accumulate p53 and undergo apoptosis. This variation provides one explanation for the tissue specificity of tumor suppression by p53. It also indicates that the role of apoptosis in the response of tumors to therapy varies significantly among tumor types.     

Survivin stable knockdown by siRNA inhibits tumor cell growth and angiogenesis in breast and cervical cancers

Increased resistance to apoptosis is a hallmark of many tumor cells. Survivin, a member of IAP family protein, is expressed in many human cancers and plays an important role in protecting cells from apoptosis. Here we show that vector-based small interfering RNAs (siRNA) stably knockdown survivin expression in several cancer cell lines, leading to increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin or TNF-alpha. The apoptotic susceptibility was dependent on divergent levels of survivin expression. The stable transfectants exhibited abnormal morphology, suppressed cell growth, enhanced spontaneous apoptosis and cell cycle hindrance. Furthermore, in nude mice xenografts of survivin-positive tumors, cells expressing survivin-targeted siRNAs exhibited decreased tumor formation and reduced angiogenesis. Results from these studies: (1) provide direct evidence that intracellular silencing of survivin by siRNA sensitizes human tumor cells to apoptosis; (2) define survivin as a promising molecular target for cancer therapy; and (3) suggest the potential applicability of survivin-targeted siRNA for treating human tumors, probably in combination with chemotherapy.     

Nonradioactive iodide effectively induces apoptosis in genetically modified lung cancer cells

We assessed a nonradioactive approach to induce apoptosis in non-small cell lung cancer by a novel iodide uptake and retention mechanism. To enhance tumor apoptosis, we transduced non-small cell lung cancer cells with retroviral vectors containing the sodium iodide symporter (NIS) and thyroperoxidase (TPO) genes. Expression of NIS and TPO facilitated concentration of iodide in tumors. As a consequence of the marked increase in intracellular levels of iodide, apoptosis was seen in >95% of NIS/TPO-modified lung cancer cells. Intraperitoneal injection of potassium iodide resulted in significant tumor volume reduction in NIS/TPO-modified tumor xenografts without apparent adverse effects in SCID mice. Iodide induced an increase in the level of reactive oxygen species. Iodide-induced apoptosis is sensitive to N-acetylcysteine inhibition, suggesting an important role by reactive oxygen species in this apoptotic process. In addition, iodide-induced apoptosis is associated with overexpression of CDKN1A (p21/Waf1)and down-regulation of survivin at both mRNA and protein levels. This is the first report demonstrating that a therapeutic dose of nonradioactive iodide has potent efficacy and high selectivity against lung cancer when used in combination with genetic modification of cancer cells to express the NIS/TPO genes.     

Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines

PURPOSE: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human non-small cell lung cancer (NSCLC) cell lines to gamma-radiation. EXPERIMENTAL DESIGN: The radiosensitizing effect of YM155 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation. RESULTS: YM155 induced down-regulation of survivin expression in NSCLC cells in a concentration- and time-dependent manner. A clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to gamma-radiation in vitro. The combination of YM155 and gamma-radiation induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of histone gamma-H2AX also showed that YM155 delayed the repair of radiation-induced double-strand breaks in nuclear DNA. Finally, combination therapy with YM155 and gamma-radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. CONCLUSIONS: These results suggest that YM155 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of YM155 is likely attributable, at least in part, to the inhibition of DNA repair and enhancement of apoptosis that result from the down-regulation of survivin expression. Combined treatment with YM155 and radiation warrants investigation in clinical trials as a potential anticancer strategy.     

Aerosol delivery of PEI-p53 complexes inhibits B16-F10 lung metastases through regulation of angiogenesis

Inhibition of pulmonary metastases poses a difficult clinical challenge for current therapeutic regimens. We have developed an aerosol system utilizing a cationic polymer, polyethyleneimine (PEI), for topical gene delivery to the lungs as a novel approach for treatment of lung cancer. Using a B16-F10 murine melanoma model in C57BL/6 mice, we previously demonstrated that aerosol delivery of PEI-p53 DNA resulted in highly significant reductions in the tumor burden (P < .001) in treated animals, and also lead to about 50% increase in the mean length of survival of the mice-bearing B16-F10 lung tumors. The mechanisms of this antitumor effect of p53 are investigated in this report. Here, we demonstrate that the p53 transfection leads to an up-regulation of the antiangiogenic factor thrombospondin-1 (TSP-1) in the lung tissue and the serum of the mice. Furthermore, there is a down-regulation of vascular endothelial growth factor (VEGF) in the lung tissue and serum of the B16-F10 tumor-bearing mice treated with PEI-p53 DNA complexes, compared with untreated tumor-bearing animals. In addition, staining for von Willebrand factor (vWF), a marker for the angiogenic blood vessels, revealed that p53 treatment leads to a decrease in the angiogenic phenotype of the B16-F10 tumors. Immunohistochemistry for transgene expression reveals that the PEI-p53 aerosol complexes transfect mainly the epithelial cells lining the airways, with diffuse transfection in the alveolar lining cells, as well as, the tumor foci in the lung tissue. There was also some evidence of apoptosis in the lung tumor foci of animals treated with p53. The data suggest that aerosol delivery of PEI-p53 complexes leads to inhibition of B16-F10 lung metastases, in part by suppression of angiogenesis.     

口服VEGFR2 DNA疫苗抗小鼠Lewis肺癌血管生成的实验研究

背景与目的血管内皮生长因子(VEGF)及其主要受体血管内皮生长因子受体-2(VEGFR2)在肿瘤新生血管和肿瘤基质形成过程中起着重要作用。本研究的目的是观察口服VEGFR2 DNA疫苗抗C57BL/6小鼠Lewis肺癌皮下移植瘤生长的作用,并探讨其可能的作用机制。方法将重组DNA疫苗对小鼠进行免疫,通过观察小鼠Lewis肺癌皮下移植瘤大小,记录各组小鼠离体肿瘤湿重,检测移植瘤微血管密度(MVD)及血液CD3 、CD8 T细胞水平,评价重组疫苗的抑瘤作用。结果疫苗组、空质粒组、生理盐水组的MVD分别为1.75±1.07、6.89±2.52、7.57±3.75,肿瘤湿重分别为(2.05±1.32)、(4.83±1.47)、(5.12±1.02)g,疫苗组与其它两组比较,差异均有统计学意义(P<0.05)。接种肿瘤后,疫苗组CD3 T细胞仍维持较高水平,其它两组明显下降(P<0.05);疫苗组CD8 T细胞水平较其它两组明显升高(P<0.05)。结论口服VEGFR2DNA疫苗对小鼠Lewis肺癌皮下移植瘤的生长具有较强的抑制作用。该疫苗可能是通过杀伤肿瘤内皮细胞、抑制血管生成而起到抗肿瘤生长的作用。     

Differences in the effects of host suppression on the adoptive immunotherapy of subcutaneous and visceral tumors

A syngeneic transplantable sarcoma induced in C57BL/6 mice, MCA 105, was used in studies to examine host suppression on the adoptive immunotherapy of established intradermal and experimentally induced pulmonary and hepatic metastases. Fresh immune splenocytes were generated from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum. The adoptive immunotherapy of intradermal MCA 105 tumor with immune cells required prior immunosuppression of the recipient by sublethal irradiation with 500 R or T-cell depletion. The effect of whole-body sublethal irradiation appeared to eliminate a systemic host suppression mechanism, since partialbody irradiation involving the tumor-bearing area did not permit successful immunotherapy. Host irradiation was not required to achieve successful immunotherapy of experimentally induced pulmonary or hepatic metastases. In nonirradiated recipients bearing both intradermal and pulmonary tumors, host suppression did not affect the function of transferred immune cells to induce regression of pulmonary metastases. Thus, suppression of adoptive immunotherapy appears to be relevant to tumors confined to the skin and subcutaneous tissue but not to tumor in visceral sites, such as the lung and liver.     

Inhibitory effects of heparin plus cortisone acetate on endothelial cell growth both in cultures and in tumor masses

A combination of heparin and cortisone acetate significantly inhibited both embryonic angiogenesis and the tumor growth of Lewis lung carcinoma (3LL) transplanted into C57BL/6 mice, although each of these agents used alone affected neither angiogenesis nor tumor growth. On the other hand, this combination neither decreased the number of metastatic foci in the lung nor prolonged the survival time of mice with 3LL. All tumor-bearing mice died of hemothorax due to pulmonary metastases. Cortisone acetate by itself increased metastasis, and addition of heparin did not affect accelerated metastasis. Because an antiangiogenic activity appears independent of metastasis acceleration by cortisone acetate, the use of steroids other than cortisone acetate having no metastasis-promotion effect should be required for an antiangiogenic tumor therapy in the presence of heparin. Heparin plus cortisone acetate prevented the DNA synthesis of cultured vascular endothelial cells but not that of cultured 3LL cells. Additionally, oral administration of this combination decreased the [3H]thymidine labeling of endothelial cells of tumor blood vessels prior to the suppression of tumor growth. The specific inhibition of the growth of endothelial cells by heparin plus cortisone acetate was revealed in both the in vitro and the in vivo tests.     

Enhanced metastatic dissemination to multiple organs by melanoma and lymphoma cells in timp-3-/- mice

Identifying versatile inhibitors of metastasis that operate in multiple sites against distinct cancer cell types is important for designing novel therapeutics for metastasis. We show that multiple tissues of timp-3-/- mice are more susceptible to metastatic colonization. Overall, a 5-14-fold increase in liver and kidney colonization occurred by EL-4 lymphoma cells, and a twofold increase upon targeting B16F10 melanoma cells to the bone or lung of timp-3-/- mice. There was a general lack of macrophage or neutrophil localization to metastases in the liver, kidney and lung, and of osteoclasts to bone in both genotypes. Analysis of lung showed that proliferation or angiogenesis were unaltered within the metastatic colonies. Lung-trap assays revealed that initial tumor cell trapping was similar in the lung vasculature of timp-3-/- and wild-type mice. However, more tumor cells were found in timp-3-/- lungs at 48 and 96 h after tumor cell injection indicating more efficient extravasation and initial proliferation. Activation of pro-MMP-2 was greater in timp-3-/- lungs at these time points. These data demonstrate TIMP-3 functions to inhibit metastatic dissemination of diverse cancer cells to multiple organs. TIMP-3 regulates MMP-2 activation to limit tumor cell extravasation and subsequent colonization of the lung, without augmenting inflammatory cell response.     

Triplex-forming oligodeoxynucleotides targeting survivin inhibit proliferation and induce apoptosis of human lung carcinoma cells

Survivin is expressed in most cancers but is undetectable in differentiated adult cells, and plays an important role both in the suppression of apoptosis and mitotic spindle checkpoint; thus it has attracted great interest as a potential drug target. In this study, we investigated the antigene and antiproliferative effects of triplex-forming oligodeoxynucleotides (TFO) targeting survivin in human lung carcinoma A549 cells. Survivin-specific TFOs form stable triplexes under physiological conditions as tested by electrophoretic mobility shift assays. Treatment of A549 cells with survivin-specific but not control TFOs at a concentration of 400 nM in the presence of uptake-enhancing liposome significantly reduced survivin protein level, inhibited cell proliferation, and induced cell apoptosis as demonstrated by immunoblot, cell number counting, and Annexin V-staining. Moreover, we found that the triplex-forming potential of TFOs measured in vitro does not necessarily correlate with the ability of TFOs to affect expression of a targeted gene in vivo. Our results indicate that targeting survivin is a promising alternative strategy for the development of novel anticancer therapeutics.